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Prokaryotic expression engineering bacteria for producing human recombinant interleukin-15 and its purifying method

A technology of interleukin and recombinant vector, applied in the field of prokaryotic expression engineering bacteria and purification of recombinant protein, can solve problems such as leakage

Inactive Publication Date: 2005-06-01
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, 6 times the dose of IL-15 can cause toxic side effects such as pulmonary vascular leakage

Method used

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  • Prokaryotic expression engineering bacteria for producing human recombinant interleukin-15 and its purifying method
  • Prokaryotic expression engineering bacteria for producing human recombinant interleukin-15 and its purifying method
  • Prokaryotic expression engineering bacteria for producing human recombinant interleukin-15 and its purifying method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Total synthesis of full-length human interleukin-15 mature gene

[0065] according to figure 1 The principle of fully synthesizing human interleukin-15 mature gene by PCR method is shown. The two parts of the four pairs of primers are complementary to each other, and each uses the other as a template to synthesize DNA fragments under the action of Taq enzyme. Similarly, the synthesized DNA fragments can also be partially complementary to each other to synthesize longer DNA fragments. Therefore, short fragments, sub-full-length and full-length human interleukin-15 mature gene DNA sequences can be synthesized by stepwise bridge splicing. The 3' ends of primers L1s, L2s, L3s, and L4s are complementary to the 3' ends of primers L1a, L2a, L3a, and L4a, and the 5' ends of primers L2s, L3s, and L4s are paired with the 5' ends of primers L1a, L2a, and L3a, respectively end complementary pairing. The small primers are used in the second and third rounds of PCR amplification...

Embodiment 2

[0067] Construction of recombinant expression vector

[0068] The technical route for the construction of recombinant expression vectors is as follows: Figure 7 and Figure 8 shown. Firstly, the full-length mature human interleukin-15 gene was spliced ​​by polymerase chain reaction (PCR), and enzyme cutting sites were added at its 3' and 5' ends, and primers such as figure 2 and Figure 4 shown. Use pBV 220 To construct the recombinant expression vector, the restriction sites of PstI and EcoRI, namely CTGCAG and GAATTC, were added to the 3' and 5' ends of rhIL-15, respectively. use pET 42a To construct a recombinant expression vector, add XhoI and NdeI restriction sites, namely CTCGAG and CATATG, to the 3' and 5' ends of rhIL-15, respectively. The full-length human interleukin-15 mature gene with restriction sites was loaded into pGEM-T plasmid and sequenced, and the pGEM-T plasmid with full-length human interleukin-15 mature gene was encoded with PstI and EcoRI Doubl...

Embodiment 3

[0070] Engineering bacteria BL 21 star TM (DE3)plysS / pBV 220 - hIL-15 and BL 21 star TM (DE3)plysS / pET 42 - Obtaining, screening and identification of hIL-15

[0071] Step 1. Screen engineering bacteria with stable and high expression

[0072](1) the pBV obtained in the above-mentioned embodiment 2 220 - hIL-15 plasmid and pET 42 -hIL-15 plasmid transformed into host strain BL 21 star TM (DE3) plysS, the engineering bacterium of the present invention;

[0073] (2) pick monoclonal colony BL in solid LB agar medium plate 21 star TM (DE3)plysS / pBV 220 - hIL-15 in 4ml LB liquid medium at 28°C, 250 rpm, shake the bacteria overnight;

[0074] (3) Inoculate in 4ml LB medium at a ratio of 1:100, shake the bacteria to OD at 28°C, 250 rpm 600 When the value is 0.4-0.6, quickly raise the temperature to 42°C to induce expression, continue to shake the bacteria for 4-5 hours, and recover the bacteria.

[0075] (4) As for the engineering bacteria BL 21 star TM (DE3)ply...

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Abstract

The present invention relates to one kind of prokaryotic system expression and purification technology consists of being used in large scale production of recombinant human interleukin-15. specifically, the technology of the present invention includes one kind of engineering bacterian and its completely new expression and purification technology. The engineering bacterium contains one completely new recombinant expression vector with one completely new synthesized DNA segment.

Description

technical field [0001] The invention relates to two prokaryotic expression engineering bacteria for human recombinant interleukin-15 (recombinant human interleukin-15, rhIL-15) and the recombinant protein purification technology thereof. More specifically, the engineered bacterium of the present invention contains a brand-new recombinant expression vector, in which there is a newly synthesized DNA segment. The invention also relates to the induced expression and purification technology of the recombinant protein. Background technique [0002] Interleukin (IL)-15 is a cytokine of about 12-14kD discovered by Grabstein et al. in 1994. With the deepening of its research, people have updated the structure and function of IL-15 understanding. Similar to most cytokines, IL-15 can play a role in the body's normal immune response, such as promoting the development of T cells, B cells, and natural killer (NK) cells. IL-15 can also exert broad and pleiotropic effects on various tiss...

Claims

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Application Information

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IPC IPC(8): C07K1/20C07K14/54C12N1/20C12N15/24C12N15/63C12N15/70
Inventor 唐峰何维
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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